Inborn errors of mitochondrial fatty acid metabolism are a heterogeneous group of disorders, the first of which was described only 20 years ago. The acyl-CoA dehydrogenases (ACDs) are a family of highly conserved enzymes which catalyze the first intra-mitochondrial step in beta- oxidation with maximum specificities for various chain length fatty acyl-CoA substrates. Deficiency of short chain acyl-CoA dehydrogenase (SCAD) has presented particular diagnostic problems. Excretion of ethylmalonic acid (EMA) in the urine is non-specific and does not reliably allow diagnosis of SCAD deficiency. One patient has been confirmed to have an SCAD deficiency at the molecular level, but other claims of this deficiency have been based solely on enzyme analysis for diagnosis. More recent data from our lab have suggested that at least some of these patients have reduced but detectable levels of SCAD. A number of polymorphic variants of SCADS have recently been identified by our collaborator Dr. Niels Gregersen which occur in high frequency in individuals of northern European ancestry. While most individuals carrying these alleles appear to show no obvious clinical phenotype, two of them are greatly over represented in a population identified on the basis of excess EMA excretion in a variety of clinical settings. The long range goal of this project is to better understand the metabolism of short chain acyl-CoAs in humans and to characterize deficiency of SCAD at the clinical, biochemical, structural and molecular level. An integrally related goal is to understand the role of the variant SCADS in the development of disease. Specific Aim 1 is to identify molecular defects responsible for causing SCAD deficiency, to characterize the effects of mutations in SCAD on enzyme function, and to describe the clinical phenotype associated with SCAD deficiency. Specific Aim 2 is to characterize the in vivo and in vitro function of the polymorphic variant SCADS and their clinical significance. Specific Aim 3 is to utilize knowledge of the crystal structure of the various ACDs to identify the amino acid residues and motiffs important in determining the specificity of members of this gene family for short chain acyl-CoA substrates.